Method for detecting dna adducts in saliva

Abstract

A method of detecting DNA adducts in saliva is disclosed herein. The method comprises the following steps: providing a salivary DNA; adding at least one isotope-labeled internal standard and a plurality of enzymes into the salivary DNA to hydrolyze the salivary DNA into a plurality of nucleosides; extracting the plurality of nucleosides by using a solid-phase extraction column; and detecting and quantifying at least one DNA adduct in the plurality of extracted nucleosides by utilizing a stable isotope dilution nano-flow liquid chromatography-nanospray ionization tandem mass spectrometry (LC-NSI/MS/MS).

1 . A method of detecting DNA adduct in saliva comprising:
 providing a salivary DNA (deoxyribonucleic acid);   adding at least one isotope-labeled internal standard and a plurality of enzymes into the salivary DNA to hydrolyze the salivary DNA into a plurality of nucleosides, wherein the isotope-labeled internal standards are [ 15 N 5 ]AdG, [ 15 N 5 ]CdG, [ 15 N 5 ]εdAdo, [ 15 N 3 ]εdCyd and [ 13 C 1 , 15 N 2 ],1,N 2 -εdGuo;   extracting the nucleosides by utilizing a solid-phase extraction column; and   detecting and quantifying at least one DNA adduct having one of 1,N 2 -propano-2′-deoxyguanosine derived from acrolein (AdG); 1,N 2 -propano-2′-deoxyguanosine derived from crotonaldehyde (CdG): 1,N 6 -etheno-2′-deoxvadenosine (εdAdo); 3,N 4 -etheno-2′-deoxvcytidine (εdCyd) and 1,N 2 -etheno-2′-deoxyguanosine (1,N 2 -εdGuo) with a quantification limit of 0.1 picogram, 0.5 picogram, 0.1 picogram, 0.5 picogram and 0.5 picogram, respectively, in the plurality of extracted nucleosides by utilizing a stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (LC-NSI/MS/MS).   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of detecting DNA adduct in saliva as recited in  claim 1 , wherein the plurality of enzymes include micrococcal nuclease, phosphodiesterase II, adenosine deaminase and alkaline phosphatase. 
     
     
         6 . The method of detecting DNA adduct in saliva as recited in  claim 1 , wherein the salivary DNA is at least 25 micrograms. 
     
     
         7 . The method of detecting DNA adduct in saliva as recited in  claim 1 , wherein a spray voltage of the stable isotope dilution nanoflow LC-NSI/MS/MS is 1.3 to 2.0 kV. 
     
     
         8 . The method of detecting DNA adduct in saliva as recited in  claim 1 , wherein a source temperature of the stable isotope dilution nanoflow LC-NSI/MS/MS is 200 to 300 degrees Celsius. 
     
     
         9 . (canceled)